Self-assembly and mineralization of peptide-amphiphile nanofibers

ABSTRACT

Peptide-amphiphilic compositions capable of self-assembly into useful nanostructures.

This application is a divisional of U.S. patent application Ser. No.10/294,114, filed Dec. 6, 2004, which claims priority from U.S.Provisional Application No. 60/333,074, filed Nov. 14, 2001, thecontents of both of which are incorporated herein by reference in theirentireties.

The United States government has certain rights to this inventionpursuant to Grant Nos. DE-FG02-00ER45810/A001, DMR9996253 andF49620-00-1-0283/P01 from, respectively, the DOE, NSF and AFOSR-MURI toNorthwestern University.

BACKGROUND OF THE INVENTION

Self-assembly and biomineralization are used for fabrication of manycomposite materials. Bone tissue is a particularly complex example ofsuch a composite because it contains multiple levels of hierarchicalorganization (S. Weiner, H. D. Wagner, Annu. Rev. Mater. Sci. 28,271-298 (1998)). At the lowest level of this hierarchy is theorganization of collagen fibrils with respect to hydroxyapatite (HA)crystals. Collagen fibrils are formed by self-assembly of collagentriple helices while the HA crystals grow within these fibrils in such away that their c-axes are oriented along the long axes of the fibrils(W. Traub, S. Weiner, Proc. Nat. Acad. Sci. 86, 9822-9826 (1989)). Thepreparation of any material with structure on the nanoscale is achallenging problem. Fabrication of materials that resemble bone, evenat the lowest level of hierarchical organization, is even more difficultbecause it involves two dissimilar organic and inorganic nanophases thathave a specific spatial relationship with respect to one another. Oneapproach, using an artificial system, has been to prepare an organicnanophase designed to exert control over crystal nucleation and growthof the inorganic component.

The controlled nucleation and growth of crystals from organic templateshas been demonstrated in in vitro experiments and in a number of naturalbiomineralizing systems (S. Mann, J. P. Hannington, R. J. P. Williams,Nature 324, 565-567 (1986); D. D. Archibald, S. Mann, Nature 364,430-433 (1993); S. L. Burkett, S. Mann, Chem. Commun. 321-322 (1996); S.I. Stupp, P. V. Braun, Science 277, 1242-1248 (1997); J. Aizenberg, A.J. Black, G. M. Whitesides, Nature 398, 495-498 (1999); S. R. Whaley, D.S. English, E. L. Hu, P. F. Barbara, A. M. Belcher, Nature 405, 665-668.(2000); L. Addadi, S. Weiner, Angew. Chem., Int. Ed. Engl. 31, 153-169(1992); S. Mann, J. Chem. Soc., Dalton Tran. 3953-3961 (1997); S.Weiner, L. Addadi, J. Mater. Chem. 7, 689-702 (1997)). These studies ontemplated crystal growth suggest that nucleation occurs on surfacesexposing repetitive patterns of anionic groups. Anionic groups tend toconcentrate the inorganic cations creating local supersaturationfollowed by oriented nucleation of the crystal. Many groups haveinvestigated the preparation of bone-like materials using threedimensional organic substrates such as poly(lactic acid), reconstitutedcollagen and many others, and some studies shows a similar correlationbetween the crystallographic orientation of hydroxyapatite when theorganic scaffold is made from reconstituted collagen (G. K. Hunter, H.A. Goldberg, Biochem. J. 302, 175-179 (1994); G. M. Bond, R. H. Richman,W. P. McNaughton, J. Mater. Eng. Perform. 4, 334-345 (1995); J. -H.Bradt, M. Mertig, A. Teresiak, W. Pompe, Chem. Mater. 11, 2694-2701(1999); N. Ignjatovic, S. Tomic, M. Dakic, M. Miljkovic, M. Plavsic, D.Uskokovic, Biomaterials 20, 809-816 (1999); F. Miyaji, H.-M. Kim, S.Handa, T. Kokubo, T. Nakamura, Biomaterials 20, 913-919 (1999); H. K.Varma, Y. Yokogawa, F. F. Espinosa, Y. Kawamoto, K. Nishizawa, F.Nagata, T. Kameyama, J. Mater. Sci.: Mater. Med. 10, 395-400 (1999); A.Bigi, E. Boanini, S. Panzavolta, N. Roveri, Biomacromolecules 1, 752-756(2001); M. Kikuchi, S. Itoh, S. Ichinose, K. Shinomiya, J. Tanaka,Biomaterials 22, 1705-1711 (2001)). However, such results have neverbeen demonstrated in a pre-designed and engineered self-assemblingmolecular system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. In accordance with this invention: a) Chemical structure of apreferred peptide amphiphile, highlighting one or more structuralfeatures thereof. Region 1 may comprise a long alkyl tail that conveyshydrophobic character to the molecule and combined with the peptideregion makes the molecule amphiphilic. Region 2 may comprise one or more(four consecutive, shown) cysteine residues which when oxidized may formdisulfide bonds to polymerize the self-assembled structure. Region 3 maycomprise a flexible linker region of one or more glycine residues,preferably three, or functionally similar such residues or monomers, toprovide the hydrophilic head group flexibility from the more rigidcrosslinked region. Region 4 may comprise a single phosphorylated serineresidue which is designed to interact strongly with calcium ions andhelp direct mineralization of hydroxyapatite. Region 5 may comprise acell adhesion ligand RGD. b) Molecular model of an illustrated PAshowing the overall conical shape of the molecule going from the narrowhydrophobic tail to the bulkier peptide region. c) Schematic showing theself-assembly of PA molecules into a cylindrical micelle.

FIG. 2. a) Negative stain (phosphotungstic acid) TEM of self-assemblednanofibers before covalent capture. Fibers are arranged in ribbon-likeparallel arrays. b) Vitreous ice cryo-TEM of the fibers reveals thediameter of the fibers in their native hydrated state to be 7.6±1 nm. c)Positive stain (uranyl acetate) TEM of the self-assembled nanofibersafter oxidative cross-linking showing electron dense regions due to thestain that localized on the periphery of the fibers. d) Thin section TEMof positively stained (uranyl acetate) nanofibers after oxidativecross-linking and embedding in epoxy resin. Two fibers are observed incross-section (arrows) clearly showing the lack of staining in theinterior of the fiber.

FIG. 3. a) TEM micrographs of the unstained, cross-linkedpeptide-amphiphile fibers incubated for 10 min in CaCl₂ and Na₂HPO₄solution. The fibers arranged in bundles are visible due to the highconcentration of inorganic ions on their surface. b) After 20 minutesforming HA crystals (arrows) are observed in parallel arrays on some ofthe PA fibers. c) After 30 minutes mature HA crystals (arrows)completely cover the PA fibers. d) Electron diffraction pattern takenfrom a mineralized bundle of PA fibers after 30 minutes of exposure tocalcium and phosphate. The presence and orientation of the diffractionarcs corresponding to the 002 and 004 planes indicate preferentialalignment of the crystals with their c-axes along the long axis of thebundle. e) Plot of intensity versus inverse angstroms reveals that the002 and 004 peaks of hydroxyapatite are strongly enhanced along thepeptide-amphiphile fiber axis. f) EDS profile of mineral crystals after30 minutes of incubation reveals a Ca/P ratio of 1.67+/−0.08 as expectedfor HA.

FIG. 4. Scheme showing possible relationships between peptide-amphiphilefibers and hydroxyapatite crystals in the mineralized bundle. Arrowindicates the direction of the c-axes of the crystals.

FIG. 5. A tilt pair taken from mineralized PA fibers after 30 minutes ofincubation with calcium and phosphate demonstrating the plate shape ofthe crystals. The crystals that were “edge-on” (electron dense, narrowobjects) in the zero degree image lose contrast in the 45 degree rotatedimage (arrow 1) while the contrast of the crystals that were “face-on”in the zero degree images increase (arrow 2).

FIG. 6. a) Nonphosphorylated PA fibers after 20 minutes of incubationwith calcium and phosphate shows only amorphous mineral depositconcentrate on the fibers. b) Nonphosphorylated PA after 30 minutes ofincubation with calcium and phosphate continue to show only amorphousmineral in contrast with phosphorylated PA which shows heavycrystallization at this time point.

FIGS. 7-9. TEM micrographs for several cylindrical micelles preparedfrom PA molecules listed in Table 1. Specifically: FIG. 7, Top: Molecule#4 containing a C10 alkyl tail. Bottom: Molecule #13 containing a C22alkyl tail; FIG. 8, Top: Molecule 8 utilizing a tetra alanine sequencein place of tetra cysteine and containing a C 16 alkyl tail. Bottom:Molecule 9 utilizing a tetra alanine sequence in place of tetra cysteineand containing a C10 alkyl tail; and FIG. 9, peptide-amphiphiles withthree different peptide head groups. Top: Molecule 10 with “KGE”.Middle: Molecule 14 lacking the phosphoserine group. Bottom: Molecule 15with “IKVAV” SEQ ID NO 1.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide a nanostructured fiber-like system, or nanostructure providingother shapes such as spherical or oblate, and/or molecular componentsthereof, together with various methods for assembly and use to recreateor mimic the structural and/or functional interaction between collagenfibrils and hydroxyapatite crystals in bone or the extracellular matrixof bone prior to mineralization, thereby providing a nanostructuredapproach divergent from the prior art. It will be understood by thoseskilled in the art that one or more aspects of this invention can meetcertain objectives, while one or more other aspects can meet certainother objectives. Each objective may not apply equally, in all itsrespects, to every aspect of this invention. As such, the followingobjects can be viewed in the alternative with respect to any one aspectof this invention.

It is an object of the present invention to provide a composition whichcan be used for assembly of a molecular structure having dimensional andfunctional characteristics biomimetic with collagen fibrils.

It can also be an object of the present invention to provide ananostructured fibrous system as a template for tissue development.

It can also be an object of the present invention to provide a compositeof a mineralized nanofiber structure biomimetic with collagen fibrilsand hydroxyapatite crystals in natural bone tissue.

It can also be an object of the present invention to provide a systemfor the facile self-assembly of nanostructured fibers for use inconjunction with one or more of the preceding objectives, such fibers ascan be reversibly stabilized to promote structural integrity.

It can also be an object of the present invention to provide amethodology using fibers in a stabilized three-dimensional structure todirect and/or control mineralization and crystal growth thereon.

It can also be an object of the present invention to provide a molecularsystem for the design and engineering of specific nanofibers andcomponents thereof to target particular cell and/or mineral growth enroute to a variety of hard or soft biomimetic materials for biologicaland non-biological applications, the later including, but not limitedto, catalysis, photonics and electronics.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and its descriptions ofvarious preferred embodiments, and will be readily apparent to thoseskilled in the art having knowledge of natural biomineralizing systems.Such objects, features, benefits and advantages will be apparent fromthe above as taken into conjunction with the accompanying examples,data, figures and all reasonable inferences to be drawn therefrom.

With respect to various embodiments, the present invention comprises useof self-assembly techniques, such self-assembly as may be employed inconjunction with mineralization to prepare a nanostructured compositematerial which recreates or mimics the structural orientation andinteraction between collagen and hydroxyapatite observed in bone. Acomposite may be prepared by self-assembly, covalent capture, andmineralization of one or more peptide-amphiphile (PA) compositions. Asevident from the preceding, the PA compositions of this invention can besynthesized using preparatory techniques well-known to those skilled inthe art—preferably, by standard solid phase chemistry, with alkylationof the N-terminus of the peptide component. Mono or di-alkyl moietiesattached to the N or C termini of peptides may influence theiraggregation and secondary structure in water in both synthetic andnatural systems. As illustrated in several embodiments, a hydrophobic,hydrocarbon and/or alkyl tail component with a sufficient number ofcarbon atoms coupled to an ionic peptide can be used to create anamphiphile that assembles in water into cylindrical micelles because ofthe amphiphile's overall conical shape. (J. N. IsraelachviliIntermolecular and surface forces; 2nd ed.; Academic: London San Diego,1992). The alkyl tails pack in the center of the micelle with thepeptide segments exposed to an aqueous or hydrophilic environment. Thesecylindrical micelles can be viewed as fibers in which the chemistry ofthe peptide region is repetitively displayed on their surface.Comparably, consistent with this invention, amphiphile molecules canalso be designed to provide micelles having structural shapes that maydiffer from a fiber like appearance.

Without limitation, three structural and/or functional features can beengineered into the peptide region of a PA composition of thisinvention. First, the prepared fibers are optimally robust and, for thisreason, one or more consecutive cysteine amino acid residues—four insome embodiments—can be incorporated in the sequence for covalentcapture of supramolecular nanofibers. (D. Y. Jackson, D. S. King, J.Chmielewski, S. Singh, P. G. Schultz, J. Am. Chem. Soc. 113, 9391-9392(1991); T. D. Clark, K. Kobayashi, M. R. Ghadiri, Chem Eur J 5, 782-792(1999); Y. Y. Won, H. T. Davis, F. S. Bates, Science 283, 960-963(1999); E. R. Zubarev, M. U. Pralle, L. M. Li, S. I. Stupp, Science 283,523-526 (1999); E. A. Archer, N. T. Goldberg, V. Lynch, J Am Chem Soc122, 5006-5007 (2000); F. Cardullo, M. C. Calama, B. H. M.Snellink-Ruel, J. L. Weidmann, A. Bielejewska, R. Fokkens, N. M. M.Nibbering, P. Timmerman, D. N. Reinhoudt, Chem. Comm. 5, 367-368(2000)). Such residues can be used to form disulfide bonds betweenadjacent PA molecules upon oxidation to lock the supramolecularstructure into place. The formation of the disulfide bonds is reversibleallowing self correction of improper disulfide bonds or return to thesupramolecular structure by treatment with mild reducing agents.

With regard to a second feature, the fibers of various embodiments maybe able to nucleate the formation of HA crystals in the properenvironment. It is well known that acidic moieties play a key role inbiomineralization processes and in the formation of calcium phosphateminerals phosphorylated groups are particularly important. (G. K.Hunter, H. A. Goldberg, Biochem. J. 302, 175-179 (1994); S. Weiner, L.Addadi, J. Mater. Chem. 7, 689-702 (1997)). For example in dentin, thephosphophoryn protein family contains numerous repeats of the sequencesAsp-Ser(P)-Ser(P) and Ser(P)-Asp (A. George, L. Bannon, B. Sabsay, J. W.Dillon, J. Malone, A. Veis, N. A. Jenkins, D. J. Gilbert, N. G.Copeland, J. Biol. Chem. 271, 32869-32873 (1996)). These massivelyphosphorylated proteins are closely associated with the collagenextracellular matrix (ECM) and are known to play an important role in HAmineralization (A. Veis In Biomineralization: Chemical and BiologicalPerspectives; S. Mann, J. Webb, J. R. P. Williams, Eds.; VCH: WeinheimNew York, 1989; pp 189-222). Accordingly, at least one phosphoserineresidue can be incorporated into the peptide sequence which, after selfassembly, allows the fiber to display a highly phosphorylated surfacefunctionally biomimetic to a long peptide segment. This, in part, may beused to simulate a repetitive organization of phosphate groups found inphosphophoryn proteins.

Third, with respect to one or more of the preceding embodiments orothers within the scope of this invention, it would be beneficial forbiomedical applications to provide fibers promoting surface adhesion andgrowth of cells. Another collagen associated protein, fibronectin,contains the sequence Arg-Gly-Asp (RGD). As this sequence has been foundto play an important role in integrin-mediated cell adhesion, an RGDsequence can also be included in preferred peptide components and/or PAcompositions, depending upon end-use application. Collectively, theseand other design principles led to preparation of a PA molecule of thetype shown in FIG. 1.

Notwithstanding the numerous embodiments provided above, broader aspectsof the present invention include a peptide amphiphile compositionhaving 1) a hydrophobic component and 2) a peptide or peptide-likecomponent further including a cell adhesion sequence. In variouspreferred embodiments, the hydrophobic component of such a compositionis of sufficient length to provide amphiphilic behavior and micelleformation in water or another polar solvent system. Typically, such acomponent may be about a C₆ or greater hydrocarbon moiety, althoughother hydrophobic, hydrocarbon and/or alkyl components could be used aswould be well-known to those skilled in the art to provide similarstructural or functional effect. Regardless, a peptide component of sucha composition may include the aforementioned RGD sequence foundespecially useful for the nanofiber mineralization described herein.

Preferred peptide components of such compositions can also include aphosphoryl-functionalized (P) residue or sequence, as described above.Inclusion of a phosphoserine residue, S(P), has been found especiallyuseful for HA mineralization. Other embodiments can include, for exampleand without limitation, a phosphotyrosine residue. The peptide componentof such compositions can also include a residue or sequence capable ofpromoting intermolecular bonding and structural stability of themicelles/nanofibers available from such compositions. A sequence ofcysteine residues can be used with good effect, providing for the facileintermolecular oxidation/reduction of the associated thiolfunctionalities.

Peptide components of this invention preferably comprisenaturally-occurring amino acids. However, incorporation of knownartificial amino acids and/or other similar monomers such ashydroxyacids are also contemplated, with the effect that thecorresponding component is peptide-like in this respect. Accordingly,such artificial amino acids, hydroxyacids or related monomers can beused to meet the spacer, phosphorylation and/or intermolecular bondingobjectives described above.

Various aspects of the present invention can be described with referenceto the peptide amphiphile illustrated in FIG. 1, but consistent withbroader aspects of this invention, other compositions can be prepared inaccordance with this invention and used for the self-assembly of fibrouscylindrical micelles and corresponding nanostructures. See, Table 1,below.

TABLE 1 N- C- PA terminus Peptide (N to C) terminus 1 C16 SEQ ID NO 2:CCCCGGGS(P)RGD H 2 C16 SEQ ID NO 3: CCCCGGGS(P) H 3 H   SEQ ID NO 4:CCCCGGGS(P)RGD H 4 C10 SEQ ID NO 5: CCCCGGGS(P)RGD H 5 C6  SEQ ID NO 6:CCCCGGGS(P)RGD H 6 C10 SEQ ID NO 7: GGGS(P)RGD H 7 C16 SEQ ID NO 8:GGGS(P)RGD H 8 C16 SEQ ID NO 9: AAAAGGGS(P)RGD H 9 C10 SEQ ID NO 10:AAAAGGGS(P)RGD H 10 C16 SEQ ID NO 11: CCCCGGGS(P)KGE H 11 C10 SEQ ID NO12: AAAAGGGS(P)KGE H 12 C16 SEQ ID NO 13: AAAAGGGS(P)KGE H 13 C22 SEQ IDNO 14: CCCCGGGS(P)RGD H 14 C16 SEQ ID NO 15: CCCCGGGSRGD H 15 C16 SEQ IDNO 16: CCCCGGGEIKVAV H 16 C16 SEQ ID NO 17: CCCCGGGS(P)RGDS H 17 C_(n )SEQ ID NO 18: LLLKK-X H 18 C_(n ) LSL-X H 19 C_(n ) LSLS-X H

It should be noted that within the particular system examined, Pas 3 and5 do not exhibit micelle formation, illustrating a certain degree ofhydrophobicity as may be useful in some embodiments for self-assembly ofsuch compositions into the nanofibers or other nanostructures of thisinvention. Depending upon desired cell or mineral growth, aphosphorylated moiety or residue may not be included (see PAs 14 and15). As discussed above, cellular adhesion or interaction may bepromoted by a particular sequence of the peptide component. Withreference to PAs 10-12 and 15, a non-RGD sequence can be utilizeddepending upon cellular target or end-use application. In particular,the IKVAV (SEQ ID NO 19) sequence has been identified in other contextsas important for neuron growth and development. The YIGSR (SEQ ID NO 20)sequence, known for a role in neuronal cell-substrate adhesion, can alsobe used. Accordingly, the amphiphile compositions of this invention caninclude a peptide component having such a sequence for correspondinguse. Other residues and/or sequences capable of promoting cell adhesion,growth and/or development are known in the art and can be used inconjunction with the present invention, such residues/sequences as canbe incorporated into the peptide components and/or PA compositions ofthis invention using available synthetic techniques or straight-forwardmodifications thereof. With respect to Table 1, it is noted that severalPA compositions do not include cysteine residues: while such a residueor peptide sequence can be used to enhance intermolecular nanofiberstability, it is not required for micelle formation in the firstinstance. Reference is made to FIGS. 7-9 for TEM micrographs of severalPA compositions identified in Table 1.

Further reference is made to Table 1 and, in particular, PA compositions17-19. Various other embodiments of this invention may comprise theresidues shown, or where optionally X may further comprise one or moreof the aforementioned residues as may be utilized for intramolecularstructural flexibility, intermolecular stability, mineralization and/orcellular interaction. Accordingly, each such composition can optionallycomprise, as desired for end-use application, one or more glycine,cysteine, phosphorylated and/or cell growth, development or adhesionresidues. In accordance with the preceding discussion, the amphiphilicnature of such compositions provides for the presence of a suitablyhydrophobic component C_(n), where n is an integer corresponding to thenumber of carbon atoms in such a component sufficient to providesufficient amphiphilic character and/or assembly structure. Withoutlimitation, various embodiments of such compositions comprise ahydrophobic component of about C₆-C₂₆. More specifically and withoutlimitation, a sequence such as that provided by PA composition 17 (or asfurther comprising residue(s) X) can be utilized to modify and/orenhance the rate of nanostructure self-assembly. Likewise, withoutlimitation, the sequences of PA compositions 18 and 19 (or as furthercomprising residue(s) X) can be used, as needed, to improve thestructural or mechanical properties of the corresponding availablenanostructured gel materials. Implementation of such peptides in the PAcompositions of this invention do not, of course, preclude use of otherresidues. For example, PA compositions 17-19 can further comprise one ormore glycine, cysteine, phosphorylated and/or cellular interactionresidues, in accordance with this invention.

Various peptide-amphiphile compositions are shown in Table 1, but areprovided only by way of illustrating one or more aspects of thisinvention. It will be understood by those skilled in the art that arange of other compositions are also contemplated. For example, thepeptide components can be varied, limited only by functionalconsiderations of the sort described herein. Accordingly, peptide lengthand/or sequence can be modified by variation in number or identity ofamino acid or substituted monomer. Further, it will be understood thatthe C-terminus of any such sequence can be construed in light ofassociated carboxylate functionality or derivative thereof. While theN-terminus of the peptides is illustrated with respect to the referencedconjugated hydrophobic and/or hydrocarbon components, it will beunderstood that such components can also be varied by length and/orcomposition, such variation limited only by the functionalconsiderations presented herein. For example, a sixteen carbon (C16)component can comprise but is not limited to a straight-chain alkylhydrocarbon. As would be understood by those skilled in the art, thestructure and/or chemistry of such a component can be varied with regardto a particular, desired functionality of an amphiphile composition orassembly thereof. Likewise, the length of such a component is limitedonly by way of the degree of hydrophobicity desired for a particularamphiphile composition and/or assembled structure, in conjunction with agiven solvent medium.

In part, the present invention also provides a sol-gel systemcomprising 1) an aqueous or polar solvent solution and/or containing oneor more of the amphiphile compositions described herein, and 2) areagent to adjust system pH. As described elsewhere herein, a system pHof less than or equal to about 4, depending on peptide identity, inducesassembly, gelation or an agglomeration of the solution with suchcompositions—in some embodiments, biomimetic nanofibers of cylindricalmicelles. Conversely, use of a suitable reagent to raise the system pHto greater than or equal to about 4 dissolves or disassociates thecompositions or nanofiber micelles. In preferred embodiments, theamphiphile composition(s) of such a system includes a peptide componenthaving residues capable of intermolecular cross-linking. The thiolmoieties of cysteine residues can be used for intermolecular disulfidebond formation through introduction of a suitable oxidizing agent.Conversely, such bonds can be cleaved by a reducing agent introduced tothe system.

Corresponding to one or more embodiments of such a material, compositionor system, the present invention can also include a nanostructuredtemplate for mineral crystal and/or cellular growth. Such a templateincludes a micelle assembly of amphiphile composition(s) of the typedescribed herein, wherein the peptide component thereof may include aresidue or sequence capable of intermolecular bond formation. Inpreferred embodiments, as described above, cysteine residues can be usedfor intermolecular disulfide bond formation. Various other preferredembodiments can further include one or more phosphorylated residues topromote crystal growth and/or mineralization, depending upon a desiredmaterial or tissue target.

In the context of biomimetic hard material or tissue, the presentinvention can also include an organo-mineral composite having ananostructured peptide amphiphile template with mineral crystalsthereon. As described above, this aspect of the present invention can beillustrated with the present amphiphile compositions, assembled asnanostructured fibers, used to nucleate and grow hydroxyapatitecrystals. In preferred embodiments, the amphiphilic compositions includepeptide components having one or more residues promoting crystalnucleation and growth. Such preferred peptide components can alsoinclude one or more residues capable of intermolecular bonding tostabilize the nanofiber template. While this inventive aspect has beendescribed in conjunction with hydroxyapatite nucleation and growth, themineral component of this composite can include other inorganiccompounds and/or oxides. Such residues, sequences or moieties are of thetype described herein, or as would otherwise be understood by thoseskilled in the art made aware of this invention.

Regardless, the c-axes of the mineral crystals of such composites arealigned with the longitudinal fiber axes, in a manner analogous to thealignment observed between collagen fibrils and HA crystals in naturalbone tissue. Accordingly, the present invention can also include amethod of using a peptide amphiphile, in accordance with this invention,to promote and control HA crystal growth. The identity of the PAcompositions used therewith is limited only by way of those structuralconsiderations described elsewhere herein. Consistent therewith and withthe broader aspects of this invention, such a method includes 1)providing an aqueous or other suitable polar medium of one or morepeptide amphiphile compositions, 2) inducing assembly thereof intocylindrical micelle structures, 3) optimally stabilizing the structureswith intermolecular bond formation, and 4) introducing to the mediumreagents suitable for the preparation of HA and crystalline growththereof on the nanofiber micelle structures. As provided elsewhereherein, one or more amphiphile compositions can be used to providefibers with a variety of cell adhesion, mineralization and/or structuralcapabilities. A combination of such compositions can be used to assemblea nanofibrous matrix with synergistic properties beneficial for aparticular crystal and/or cellular development, such compositions as mayvary according to peptide component, residue sequence, hydrophobic orhydrocarbon component and/or resulting PA or assembly configuration.

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the compositions, micelles, composites and/ormethods of the present invention, including self-assembly of apeptide-amphiphile nanofiber system, as is available through themethodologies described herein. In comparison with the prior art, thepresent structures, template designs and related methods provide resultsand data which are surprising, unexpected and contrary thereto. Whilethe utility of this invention is illustrated through the use of severalamphiphiles, biomimetic micelles and resulting organo-mineralcomposites, it will be understood by those skilled in the art thatcomparable results are obtainable with various other amphiphiles,nanofibers/micelles and/or composites, as are commensurate with thescope of this invention.

Example 1

After synthesis (see, example 10), the PA of FIG. 1 (characterized by ¹HNMR and MALDI-TOF MS: [M-H]⁻¹=1333) was treated with dithiothreitol(DTT) at a pH of 8 to reduce all cysteine residues to free thiols. Atthis pH the PA was found to be soluble in excess of 50 mg/ml in water.However, upon acidification of the solution below pH 4 the materialrapidly becomes insoluble. Solutions more concentrated than 2.5 mg/mlform birefringent gels in water that are self-supporting upon inversionof the container. Examination of the gels by cryo-TEM, which preservesthe native, hydrated state of the material, revealed a network of fiberswith a diameter of 7.6±1 nm and lengths up to several microns (FIG. 2B).Negatives were digitized in the Umax PowerLook scanner with resolution1200 dpi. The average thickness of the PA fibers was determined by twodifferent procedures. First the Fourier transform of the images andfollowing measurements of the distances between the peaks were performedin the NIH Image 1201.1262 program. The second approach applied wasaveraging technique based on the cross-correlation method, widely usedin single particle reconstruction using the EMAN 1201.1202 program. Foreach data set 1200-1300 individual boxes were selected. (E. Beniash, W.Traub, A. Veis, S. Weiner, J. Struct. Biol. 132, 212-225 (2000).Positively and negatively stained dried fibers were found to havediameters of 6.0±1 nm (FIG. 2A,C). TEM using the positive stain uranylacetate, which preferentially stains acidic groups, revealed increasedelectron density at the periphery of the fiber. (J. R. Harris, Ed.Electron Microscopy in Biology, A Practical Approach; Oxford UniversityPress: New York, 1991). Additionally, gels that were stained, embeddedin epoxy resin and sectioned for TEM, showed fibers in cross-section inwhich donut shaped patterns were observed indicating staining only onthe outer portion of the fiber (FIG. 2D). The two positive stainingexperiments of this example indicate that the hydrophobic alkyl tailspack on the inside of the fiber and the acidic moieties of the peptideare displayed on the surface of the fiber.

Example 2

The formation of the fibers was found to be concentration independentover more than three orders of magnitude (0.01 mg/ml to 50 mg/ml),however a second level of hierarchy was observed that was concentrationdependent. As the concentration of the PA was increased, a larger numberof the fibers were observed to pack into flat ribbons of fibers (FIG.2A,C).

Example 3

Examination of the self-assembled material by FT-IR revealed a bimodalamide I peak with maxima at 1658 cm⁻¹ (α-helix) and 1632 cm⁻¹ (β-sheet)along with a N—H stretching peak at 3287 cm⁻¹ indicating the formationof a hydrogen bonded structure, possibly utilizing a combination ofβ-sheet and α-helical secondary structure in the fibers (S. Krimm, J.Bandekar, Adv. Protein Chem. 38, 181-364 (1986); W. K. Surewicz, H. H.Mantsch, D. Chapman, Biochemistry 32, 389-394 (1993)). Based on theabove data the nanofibers were modeled as cylindrical micelles in whichthe alkyl tails pack on the inside of the fiber and peptide segments aredisplayed on the outside containing both β-sheet and α-helical secondarystructure. This model results in a fiber with a diameter of 8.5 nm,which is within the margin of error of our TEM measurements (FIG. 1C).

Example 4

Upon lowering the pH below 4 with HCl the material self-assembles; whenthe pH is brought back to neutrality with KOH it disassembles. This pHtriggered self-assembly and gelation allows the material to respond toits environment and thus may have applications in controlled release ofmolecules (W. A. Petka, J. L. Hardin, K. P. McGrath, D. Wirtz, D. A.Tirrell, Science 281, 389-392 (1998)).

Example 5

Following self-assembly of PA molecules into fibers, the cysteine thiolgroups were oxidized by treatment with 0.01M iodine. Examination of thematerial by TEM reveals that the fibers remain intact (FIG. 2C,D). Theseoxidized PA fibers were found to be stable to alkaline solutions (pH 8)for months while fibers which had not been oxidized would disassemblewithin minutes. Based on the length of the fibers revealed by TEM andthe present model for molecular organization of fibers, the resultingpolymer has a molecular weight of roughly 2×10⁸ daltons. Importantly,the covalent capture process of this invention can be easily reversed bytreating the fibers with a mild reducing agent such as DTT. Afterreduction the fibers regain their pH sensitivity and rapidly disassembleat pH 8. Together with the controlled self-assembly, reversiblecross-linking provides a highly dynamic system which can interconvertbetween discrete molecules, self-assembled supramolecular fibers,covalently captured polymeric fibers and back again to single smallmolecules depending solely on the environment in which the material isplaced.

Example 6

To investigate the mineralization properties of PA nanofibers of thisinvention, the material was assembled and mineralized directly on aholey carbon coated TEM grid—to allow study of the dynamics of themineralization process while minimizing artifacts of TEM samplepreparation. To prepare samples, a drop of aqueous PA (1 mg/ml) wasmounted on the holey grid and the self-assembly of the PA was induced inan atmosphere of HCl vapor. The grids were immersed in aqueous iodine tooxidize the cysteine thiol groups to disulfides. After rinsing withdistilled water, the PA coated grids were treated with 5 μl of 10 mMCaCl₂ on one side and 5 μl of 5 mM Na₂HPO₄ on the other side. The twosolutions are able to mix only by passing through the holes in thecarbon support. Grids were examined by TEM at different time intervals(FIG. 3) and after 10 minutes inorganic material was observed to beconcentrated around the fibers thus increasing their contrast (acrystalline phase was not detected at this time by electrondiffraction). At 20 minutes the fibers begin to be covered withcrystalline mineral, although significant amorphous material remains.After 30 minutes plate shaped polycrystalline mineral is visiblethroughout the surface of the fibers (FIG. 3C and FIG. 5). The mineralwas analyzed by EDS (Energy Dispersion X-Ray Fluorescence Spectroscopy)which revealed a Ca/P ratio of 1.67±0.08 which is consistent with theformation of HA with a formula of Ca₁₀(PO₄)₆(OH)₂ (FIG. 3F).

Example 7

As controls for the experiment of example 6, carbon coated TEM gridswere treated as above but without PA fibers. In this case no mineraldeposit was found on the grids. In a second control, a PA was preparedin which phosphoserine was replaced by serine and treated as above withcalcium and phosphate. The nonphosphorylated fibers were observed by TEMafter 20 and 30 minutes of incubation and in both cases an amorphousmineral deposit around the fibers was observed, but crystals did notform within this time frame (FIG. 6).

Example 8

In order to discern the relative orientation of the HA crystals withrespect to PA fibers, several samples of example 6, in which isolatedmineralized bundles of PA fibers could be observed, were analyzed byelectron diffraction. In all cases preferential alignment of the HAcrystallographic c-axis with the PA fiber long axis was observed. (FIG.3D,E).

Example 9

Mineralization experiments show that PA fibers of this invention areable to nucleate hydroxyapatite on their surfaces. Negatively chargedsurfaces can promote mineralization by establishing local ionsupersaturation (S. Weiner, L. Addadi, J. Mater. Chem. 7, 689-702(1997)). Particularly, the two acidic aminoacids phosphoserine andaspartic acid used here are abundant in the proteins of mineralizedtissues proven to initiate crystal growth (L. Addadi, S. Weiner, Proc.Natl. Acad. Sci. 82, 4110-4114 (1985); G. Falini, S. Albeck, S. Weiner,L. Addadi, Science 271, 67-69 (1996); A. George, L. Bannon, B. Sabsay,J. W. Dillon, J. Malone, A. Veis, N. A. Jenkins, D. J. Gilbert, N. G.Copeland, J. Biol. Chem. 271, 32869-32873 (1996); S. Weiner, L. Addadi,J. Mater. Chem. 7, 689-702 (1997)). The fact that the fibers gain extraelectron density prior to formation of the crystalline phase suggeststhe above mechanism may be utilized in our system. More surprising isthe observation that the c-axes of the HA crystals are co-aligned withlong axes of the fibers (FIG. 4). This fact implies that the orientationof crystalline nuclei and the subsequent crystal growth are not randombut are controlled by the PA micelles. The exact mechanism of thiscontrol is not clear, however in similar systems such control is gainedby specific arrangement of acidic groups that promote growth of thecrystals in a particular orientation by an epitaxial mechanism (S. Mann,J. P. Hannington, R. J. P. Williams, Nature 324, 565-567 (1986); S.Weiner, L. Addadi, J. Mater. Chem. 7, 689-702 (1997); J. Aizenberg, A.J. Black, G. M. Whitesides, Nature 398, 495-498 (1999)). An analogousmechanism may be employed with PA fibers. Previous in vitro studiesshowing oriented crystal growth from organic templates were done mainlyin two dimensional systems. The results of this example show for thefirst time oriented crystal growth in a synthetic fiberous organicsubstrate.

Example 10

Several representative peptide-amphiphiles of this invention wereprepared by manual solid phase peptide synthesis starting from 0.5mmoles of an FMOC-Asp(tBu)-WANG resin. Deprotection of the initial andsubsequent FMOC groups and was accomplished by double treatment with 15ml of 30% piperidine in DMF for 2 minutes and 7 minutes. The resin wasthen washed with 15 ml of DMF 5 times. With the exception of cysteinederivatives, amino acids (4 equivalents) were preactivated with HBTU(3.95 equivalents) and DiEA (6 equivalents) in 10 ml of DMF for twominutes. The activated solution was then added to the deprotected resinand allowed to shake for 30 minutes after which a small sample wasremoved and analyzed with ninhydrin to test for completeness of thereaction. Failed reactions were recoupled in an identical fashion.Cysteine was coupled via the FMOC-Cys(Trt)-OPfp activated ester with theaddition of 1 equivalent of DiEA in order to avoid suspectedepimerization found using the standard FMOC-Cys(ACM)-OH derivative withthe conditions described above. Other amino acid derivatives usedincluded FMOC-Gly-OH, FMOC-Arg(Pbf)-OH and FMOC-Ser(PO(OBzl)OH)-OH.Analogous derivatives available in the art may be used to couple A, S,L, K and other such residues, including those shown in Table 1.Protecting groups may vary depending on the subject amino acid residue.The final step was coupling of a suitable fatty acid or derivativethereof (e.g., palmitic acid) and was accomplished using 2 equivalentsof the acid activated with 2 equivalent of HBTU and 3 equivalents ofDiEA in 15 ml DMF for 2 minutes. The solution was allowed to react withthe resin overnight. Likewise, other reagents and starting materials ofthe sort useful in the preparation of the inventive compositions,including but not limited to those shown in Table 1, arereadily-available and known to those skilled in the art, suchreagents/starting materials as may be used for thehydrophobic/hydrocarbon and peptide (residues/monomers) components.

Cleavage and deprotection of the peptide-amphiphiles was done with amixture of TFA, water, triisopropyl silane (TIS) and ethane dithiol(EDT) in a ratio of 91:3:3:3 for three hours at room temperature. Thecleavage mixture and TFA washings were filtered into a round bottomflask. The solution was roto-evaporated and then redissolved in aminimum of neat TFA. This solution was triturated with colddiethylether. The white precipitation was collected by filtration anddried under vacuum.

Various other amphiphile compositions of this invention can be preparedin analogous fashion, as would be known to those skilled in the art andaware thereof, using the above-described known procedures and synthetictechniques or straight-forward modifications thereof depending upon adesired amphiphile composition or peptide sequence.

As demonstrated by the preceding examples, figures and data, the factthat the hydroxyapatite crystals grow on the bundles of fibers withtheir c-axes oriented along the long axes of the micelles could be ofinterest for design of new materials for mineralized tissue repair. Thisnanoscale organization resembles that of hydroxyapatite crystals inmineralized ECM in which the HA crystals also grow in parallel arrayswith their c-axes co-aligned with long axes of the organic fibers (W.Traub, S. Weiner, Proc. Nat. Acad. Sci. 86, 9822-9826 (1989)). Thisarrangement is the most important characteristic of the biomineralsbelonging to the bone family (S. Weiner, H. D. Wagner, Annu. Rev. Mater.Sci. 28, 271-298 (1998)). The organization of the collagen fibers,porosity, mineral-organic ratio vary in different members of thisfamily, yet all of them are built from the collagen fibrils containingparallel arrays of hydroxyapatite crystals (W. Traub, S. Weiner, Proc.Nat. Acad. Sci. 86, 9822-9826 (1989); S. Weiner, W. Traub, FASEB J. 6,879-885 (1992); W. J. Landis, K. J. Hodgens, J. Arena, M. J. Song, B. F.McEwen, Microsc. Res. Techniq. 33, 192-202 (1996)).

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood clearly that thesedescriptions are added only by way of example and are not intended tolimit, in any way, the scope of this invention. For instance, variouspeptide amphiphiles have been described in conjunction with specificresidues and corresponding cell adhesion, but other residues can be usedherewith to promote a particular cell adhesion and tissue growth on thenanostructures prepared therefrom. Likewise, while the present inventionhas been described as applicable to biomimetic material or tissueengineering, it is also contemplated that gels or related systems ofsuch peptide amphiphiles can be used as a delivery platform or carrierfor cells or cellular material incorporated therein. Other advantagesand features will become apparent from the claims filed hereafter, withthe scope of such claims to be determined by their reasonableequivalents, as would be understood by those skilled in the art.

1-35. (canceled)
 36. A method of using a peptide amphiphile compositionto control hydroxyapatite crystal growth, said method comprising:providing a solvent medium with at least one peptide amphiphilecomposition comprising a peptide component selected from the groupconsisting of CCCCGGGSRGD, (SEQ ID NO: 2) CCCCGGGSRGDS, (SEQ ID NO: 17)CCCCGGGSKGE, (SEQ ID NO: 11) CCCCGGGEIKVAV, (SEQ ID NO: 16) AAAAGGGSRGD,(SEQ ID NO: 9) AAAAGGGSKGE, (SEQ ID NO: 12) and GGGSRGD. (SEQ ID NO: 7)

wherein the serine residue in the peptide component is phosphorylatedand wherein a C₁₀-C₂₂ hydrocarbon component is attached to theN-terminus of the peptide component; inducing assembly of said peptideamphiphile compositions into cylindrical micelles; stabilizing saidmicelles with intermolecular covalent bond formation; and introducingreagents for the preparation of hydroxyapatite.
 37. The method of claim36, wherein micelle assembly is induced by adjusting the pH of saidmedium to less than about
 4. 39. The method of claim 36, wherein thepeptide amphiphile has the peptide component is SEQ ID NO:9 and has aC₁₆ hydrocarbon component.